Experimental Study Of Inhibitory Effects Of Centipede Aqueous Extract On Cholangiocarcinoma Cell Lines QBC939 In Vitro And In Vivo And Its Mechanisms

Objective:1.To explore the inhibitory effects of Centipede Aqueous Extract on cholangiocarcinoma Cell lines QBC939 in vitro.2. To establish the nude mice subcutaneous tumors model of QBC939 cells, The treatment groups were fed with the Centipede Aqueous Extract on the concentration of lOg/kg while the control group were fed with the Saline water. The growth of the two groups were recorded in detail.3. To analysis the proteomics difference between the treatment and the control groups4.To study the possible antitumor mechanisms of Centipede Aqueous Extract on cholangiocarcinoma cell line QBC939 subcutaneous tumors by molecular biology technology.Methods:1. The morphological and colony changes of cholangiocarcinoma cell line QBC939 were observed in different concentration of Centipede Aqueous Extract by light microscopy,and the changes of the cell structure were studied by transmission electron microscopy.2. The model the nude mice subcutaneous tumors of human cholangiocarcinoma cell QBC939 were established.12 days after Subcutaneous transplantation, the treatment group nudes were fed with the Centipede Aqueous Extract on the concentration of lOg/kg while the control group were fed with the Saline water. The changes of tumor size between the treatment and control groups were recorded in details.35 days after subcutaneous transplantation, all the nude mice were anatomized and subcutaneous tumors were observed by H.E staining.3.The protein expression differences of Cholangiocarcinoma QBC939 cell subcutaneous tumors were detected by proteomics.4.To explore the mRNA and protein levels of Bcl-2 and Bax in Cholangiocarcinoma QBC939 cell subcutaneous tumors and its possible mechanism. the samples of the experimental and control groups were detected by RT-PCR and Western blot.Results:1. The growth of cholangiocarcinoma cells QBC939 could be inhibited by Centipede Aqueous Extract. With gradually increasing concentration its anti-tumor effect was also enhanced too. Its IC50 value of 48 hours was 12.5mg/ml. and Centipede Aqueous Extract could also inhibit the colony-forming of cholangiocarcinoma cell line QBC939 cells. Electron microscopy showed that the apoptosis bodies could be seen clearly in Centipede Aqueous Extract treatment cholangiocarcinoma QBC939 cells by the concentration of 12.5mg/ml after 48h. 2. Successfully established were the nude mice subcutaneous tumors model of Cholangiocarcinoma QBC939 cells. After fed with of Centipede Aqueous Extract on the concentration of 10g/kg, Obvious different Growth rates were observed between the treatment group and control groups; Plenty of vacuolization and karyopyknosis cells were seen in experimental group by HE staining,rarely in the control group.3. Compared to 38 proteins differentially expressed significantly in Cholangiocarcinoma QBC939 subcutaneous tumors tissues, among which 27 ribosomal protein genes were down-regulated significantly, while 11 ribosomal protein genes were up-regulated. CK8,CK18及Bcl-2 were down-regulated while Galectin-1 and Galectin-7 genes were down-regulated.4. The result of RT-PCR and Western blot showed that the mRNA and protein levels of Bax were increased and those of Bcl-2 were declined in the experimental groups compared to the control groupsConclusion:1. Cholangiocarcinoma QBC939 cells could be inhibited by Centipede Aqueous Extract in vitro. With gradually increasing concentration its anti-tumor effect was also enhanced too. Its IC50 value was 12.5mg/ml.2. Successfully established were the nude mice subcutaneous tumors model of Cholangiocarcinoma QBC939 cells. Centipede Aqueous Extract with the concentration of lOg/kg might have anti-tumor effect on the nude mice subcutaneous tumors.3. There were 38 proteins differentially expressed significantly in Cholangiocarcinoma QBC939 cells subcutaneous tumors tissues, CK8,CK18 and Bcl-2 were down-regulated while Galectin-1 and Galectin-7 genes were down-regulated.4. The apoptosis mechanism of treatment group Cholangiocarcinoma cells might be that up-regulated Bax and down-regulated Bcl-2 by Centipede Aqueous Extract.